PROJECT SUMMARY Mobilization of hematopoietic stem cells (HSCs) into the peripheral blood is widely used in clinical HSC transplantation to treat many blood disorders. However, the HSC supply is not sufficient and a considerable number of healthy donors and patients fail to mobilize HSCs via the standard mobilization protocol. Thus, improved strategies for inducing HSC mobilization and promoting hematopoietic repopulation after transplantation by inducing HSC proliferation are needed to improve patient outcomes. Although acute hematopoietic demands promote HSC proliferation and mobilization, little is known about the molecular mechanisms. To better understand how these HSC behaviors are regulated, we have used pregnancy in mice as a model system. We previously found that HSC proliferation, mobilization, and extramedullary hematopoiesis are induced during pregnancy when maternal blood volume expands rapidly. These responses are dependent on HSC expression of estrogen receptor ? (ER?). Signaling through this nuclear hormone receptor can be triggered by 17?-estradiol (E2) as well as 27-hydroxycholesterol (27HC), the endogenous ER ligand that is a direct metabolite of cholesterol. Importantly, different ER ligands have different effects on ER function, leading to differential effects on gene expression. Given our finding that ER? ligands differentially induce HSC proliferation and mobilization, we hypothesize that administration of ER? ligands and modulation of specific genes downstream of ER? signaling could be used to improve collection of mobilized HSCs for transplantation as well as hematopoietic repopulation after transplantation. To test this hypothesis, we propose the following Aims. In Aim 1, we will determine the potential of 27HC-ER? signaling to improve current HSC-mobilizing methods. In conjunction with clinically-used HSC-mobilizing agents, we will administer 27HC to wild-type mice or mice with humanized hematopoietic systems, and evaluate the levels of mobilized mouse and human HSCs. In Aim 2, we will determine the potential of E2-ER? signaling to enhance hematopoietic repopulation after transplantation. We will transplant mouse or human HSCs to recipient mice and treat these recipient mice with E2 to induce HSC proliferation. We will evaluate whether the treatment has beneficial effects on HSC proliferation and hematopoietic repopulation by increasing hematopoietic progenitors after transplantation. In Aim 3, we propose to identify key effector genes of ER?-mediated induction of HSC proliferation and mobilization. We will select candidate genes by comparing gene expression profiles of HSCs in mice that have either upregulated or downregulated 27HC-ER? signaling or E2-ER? signaling. We will functionally validate their roles in HSC proliferation and mobilization in vivo by lentiviral transduction. Successful completion of this study will shed light on the molecular mechanisms of HSC proliferation and mobilization, and will lay the groundwork for developing novel interventions to harvest more mobilized HSCs for clinical transplantation and to promote hematopoietic repopulation.